TY - JOUR
T1 - A Sensitive, Quantitative Assay for Human Immunodeficiency Virus Type 1 Integration
JF - Journal of Virology
JO - J. Virol.
SP - 10942
LP - 10950
DO - 10.1128/JVI.76.21.10942-10950.2002
VL - 76
IS - 21
AU - O'Doherty, Una
AU - Swiggard, William J.
AU - Jeyakumar, Deepa
AU - McGain, David
AU - Malim, Michael H.
Y1 - 2002/11/01
UR - http://jvi.asm.org/content/76/21/10942.abstract
N2 - Quantitative methods to measure human immunodeficiency virus type 1 (HIV-1) integration promise to be important tools in dissecting the mechanisms whereby latent reservoirs of provirus are established, most notably in the resting T cells of patients receiving antiretroviral therapy. Here we describe a fluorescence-monitored, nested PCR assay that is able to quantify the relatively rare integration events that occur within these cells. Following DNA extraction, a nonkinetic preamplification step is performed with primers that bind genomic Alu elements and HIV-1 gag sequences, under conditions where primers, deoxynucleoside triphosphates, and enzyme are not limiting. This is followed by a kinetic PCR that quantitates HIV-1 long terminal repeat sequences. A T-cell-based integration standard which reflects the randomness of HIV-1 integration is also described. The assay is 10 to 100 times more sensitive than previously reported quantitative Alu PCR-based integration assays. It is specific for integration events, since no proviruses are detected in cells infected either in the presence of an integrase inhibitor or with an integrase-deficient virus. This method promises to provide important new insights into the processes underlying the accumulation and persistence of latent HIV-1 reservoirs and may eventually be useful clinically in monitoring the eradication of latent virus by novel therapies.
ER -